This application claims benefit of foreign priority from Japan application 11/107246, filed Apr. 14, 1999.
The present invention relates to a novel polypeptide derived from BMS2.4 cells, a gene thereof, a method for preparing the polypeptide and the gene, and uses thereof.
Production of blood cells is strictly regulated by various stromal elements including adhesion molecules, extracellular matrix, and cytokines. Complex interactions between stromal and hematopoietic cells are essential for the movement of hematopoietic stem/progenitor cells within or from bone marrow, for the control of production of blood cells, and for the elimination of defective and harmful cells.
Long-term bone marrow cultures are in vitro system that mirror some in vivo relationships and provide an approach to define molecular and cellular interactions that may regulate production of blood cells. Pre-B cells could be displaced from the adherent layer of long-term bone marrow cultures by addition of antibodies to very late antigen-4 (VLA-4) or vascular cell adhesion molecule-1, and these reagents completely blocked lymphopoiesis in Whitlock-Witte (W/W) cultures. Antibodies to CD44 completely blocked production of lymphoid and myeloid cells in long-term bone marrow cultures. Addition of an antibody to CD9 cause strong adhesion between myeloid and stromal cells, and blocked the production of myeloid cells in Dexter cultures. These molecules might participate in cell-cell interactions critical for adhesion and movement of maturing hematopoietic cells in bone marrow.
Extracellular matrix delivers signals for survival and/or expansion of lympho-hematopoietic cells as well as immobilizes growth factors. Binding of fibronectin to integrins augments responsiveness of hematopoietic stem/progenitors to colony-stimulating factors. In contrast, interactions between fibronectin and VLA-5 cause apoptosis in a myeloid cell line. Hyaluronan, a ligand of CD44, forms viscous and hydrated gels and facilitates cell-cell adhesion and cell migration. Thrombospondin binds to hematopoietic progenitors, and hemonectin to myeloid precursors. Matrix glycoprotein SC1/ECM2 binds to B lineage cells and enhances their growth. Osteonectin not only acts as an anti-adhesive molecule but also immobilizes platelet derived growth factor.
Proliferation of primitive hematopoietic progenitors is regulated by interacting groups of cytokines. Interleukin-3 (IL-3), IL-4, and granulocyte/macrophage-colony-stimulating factor (GM-CSF) support proliferation of progenitors that exit from the dormant state. IL-6, IL-11, IL-12, granulocyte-CSF (G-CSF), and leukemia inhibitory factor (LIF) work synergistically with IL-3, IL-4, and GM-CSF to support proliferation of multipotential progenitors from cell-cycle dormant progenitors. Stem cell factor (SCF) and flt3-ligand not only support self-renewal of hematopoietic stem cells but also function as co-factors with other cytokines in promoting differentiation and expansion of hematopoietic progenitors. On the other hand, transforming growth factor-xcex2 (TGF-xcex2), tumor necrosis factor-xcex1 (TNF-xcex1), interferon-xcex1/xcex2 (IFN-xcex1/xcex2), and IFN-xcex3 are downregulators of lympho-hematopoiesis. Growth arrest and/or apoptosis of hematopoietic cells are induced by these factors. These regulatory cytokines are typically made in extremely small quantities in hematopoietic organs. Some of them are capable of attachment to extracellular matrix, and certain other cytokines are synthesized as transmembrane as well as soluble forms.
A number of genes that may be involved in lympho-hematopoiesis have been identified by experiments using cloned stromal cell lines that were originally selected for the ability to support proliferation and/or differentiation of a particular type of hematopoietic cells. A stromal cell line, BMS2, that was established from adherent cells of long-term bone marrow cultures has been known to have capacity to support growth of pre-B cells (Pietrangeli, C. E. et al., Eur. J. Immunol., 18: 863-872, 1988). However, BMS2.4 cells, a subclone of BMS2, revealed unique characteristics that they interfered with proliferation of hematopoietic cells (Kincade, P. W. et al., Adv. Exp. Med. Biol., 292: 227-234, 1991). However, its molecular mechanism is not known. An understanding of these molecules may be informative about negative regulator circuits that can potentially limit blood cell formation under steady state. These molecules may be helpful for understanding pathogenic mechanisms of lympho-hematopoietic disorders or treating such diseases.
An objective of this invention is to provide a novel protein derived from BMS2.4 cells that interferes with the proliferation of lympho-hematopoietic cells, a gene thereof, a method for preparing them, and uses thereof.
The present inventors transfected human renal carcinoma cell strain 293T with a cDNA library derived from a mouse stromal cell line, BMS2.4, and performed expression cloning based on growth inhibitory effects of the culture supernatant of transformants on myelomonocytic leukemia cell line, WEHI3. As a result, we succeeded in isolating a gene encoding a novel protein, designated Blood Cell Growth-Inhibiting Factor (BGIF) that interfered with proliferation of WEHI3 cells in a similar manner as the culture supernatant of BMS2.4 cells. A putative BGIF protein from the isolated gene is homologous to IFN-xcex1 and IFN-xcex2, and expressed in stromal cells in bone marrow and spleen.
The present inventors prepared a recombinant BGIF protein to examine its effects on the growth of various lympho-hematopoietic cells. The recombinant BGIF protein suppressed proliferation of various (pre-)B lineage cell clones (1A9, BC7.12, BC7.7, F10, 2E8, 18-81, 7OZ/3, WEHI231, WEHI279, and SP2/0), T lineage lymphoma cell line, BW1597, and multipotent cell line EML-C1, as well as WEHI3 cells. BGIF arrests the cell cycle of WEHI3 cells at the G0/G1 phase and prolongs the G1 phase. It induces apoptosis in BC7.12 cells. It also suppressed the establishment of functional adherent layers in W/W culture.
Like type I IFNs, BGIF induced IFN regulatory factor-I utlizing IFN-xcex1/xcex2 receptors, and activated JAK2 in myelomonocytic leukocyte line.
BGIF protein and its gene of this invention are associated with the lympho-hematopoietic system, and will be a useful tool for elucidating pathogenic mechanisms of lympho-hematopoietic disorders. BGIF protein of this invention would be applicable to therapeutics for various disorders in which the protein is involved.
Examples of disorders to be treated with the BGIF protein or its gene of this invention include lymphocytoma/hematapostemia such as acute lymphocytic or myelocytic leukemia, chronic lymphocytic or myelocytic leukemia, and malignant lymphoma, collagen diseases such as rheumatoid arthritis and systemic lupus erythematosus, idiopathic thrombocytopenic purpura, etc. The protein or the gene can also be used as immunoregulators. Disorders to be treated with compounds that inhibit the activity of BGIF protein include those accompanied by hematopenia such as aplastic anemia. These compounds can also be used as immunoregulators.
This invention relates to a novel protein derived from BMS2.4 cells that inhibits proliferation of lympho-hematopoietic cells, a gene thereof, and a method for preparing the protein and the gene, and uses thereof. More specifically, it relates to:
(1) a protein that suppresses the proliferation of lympho-hematopoietic cells selected from the group consisting of:
(a) a protein comprising the amino acid sequence as set forth in SEQ ID NO: 3;
(b) a protein comprising a derivative of the amino acid sequence set forth in SEQ ID NO: 3, in which one or more amino acids are substituted, deleted, inserted, and/or added; and
(c) a protein encoded by a DNA hybridizing with the DNA comprising the nucleotide sequence as set forth in SEQ ID NO: 1,
(2) the protein according to (1), wherein the lympho-hematopoietic cells are selected from the group consisting of B lineage cell line, 1A9, BC7.12, BC7.7, F10, 2E8, 18-81, 7OZ/3, WEHI231 and SP2/0, T lineage lymphoma cell line BW1597, and myelomonocytic leukemia cell line WEHI3,
(3) a DNA encoding the protein according to (1),
(4) the DNA of (3) comprising the coding region of the nucleotide sequence as set forth in SEQ ID NO: 1,
(5) a vector comprising the DNA of (3),
(6) a host cell retaining the vector of (5),
(7) a method for preparing the protein of (1), the method comprising culturing the host cell of (6) and recovering recombinant proteins expressed in the cell from the cultured cell or culture supernatant thereof,
(8) an antibody to the protein according to (1),
(9) a peptide fragment of the protein according to (1),
(10) a DNA specifically hybridizing with the DNA comprising the nucleotide sequence as set forth in SEQ ID NO: 1 and comprises at least 15 nucleotides,
(11) a method for screening a compound binding to the protein according to (1), the method comprising
(a) contacting the protein of (1) or its fragment with a test sample,
(b) detecting the binding activity of the test sample to the protein or its fragment, and
(c) selecting a compound that binds to the protein or its fragment,
(12) a compound that is isolable by the method according to (11) and binds to the protein of (1),
(13) a method for screening a compound that interferes with the activity of the protein according to (1) to suppress proliferation of lympho-hematopoietic cells, the method comprising
(a) contacting the protein according to (1) with lympho-hematopoietic cells in the presence of a test sample,
(b) detecting the proliferation of the cells, and
(c) selecting a compound that interferes with the activity of the protein according to (1) to suppress proliferation of lympho-hematopoietic cells as compared with a control where the detection is performed in the absence of the test compound,
(14) the method according to (13), wherein said lympho-hematopoietic cells are selected from the group consisting of B lineage cell lines, 1A9, BC7.12, BC7.7, F10, 2E8, 18-81, 7OZ/3, WEHI231 and SP2/0, T lineage lymphoma cell line BW1597, and myelomonocytic leukemia cell line WEHI3,
(15) a compound isolable by the method according to (13), which interferes with the activity of the protein according to (1) to suppress proliferation of lympho-hematopoietic cells,
(16) a pharmaceutical composition comprising the protein according to (1) as an active ingredient,
(17) the pharmaceutical composition according to (16), wherein the composition is for treating lympho-hematopoietic disorders, and
(18) a pharmaceutical composition comprising the compound according to (12) as an active ingredient.
Herein, xe2x80x9clympho-hematopoietic cellsxe2x80x9d mean matured or precursor cells of erythrocytes, leukocytes or platelets.